scholarly journals Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1299 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Yuji Tokunaga ◽  
Ariel Afek ◽  
Hiroki Takahashi ◽  
Nobuo Shimamoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Thermal Fluctuations ◽  
A Genome ◽  
Sequence Elements ◽  
Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.

scholarly journals Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

2020 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Yuji Tokunaga ◽  
Ariel Afek ◽  
Hiroki Takahashi ◽  
Nobuo Shimamoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Thermal Fluctuations ◽  
A Genome ◽  
Sequence Elements ◽  
Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences enriched with thymine bases represent the signal inducing abortive transcription. On the other hand, certain repetitive sequence elements broadly embedded in promoter regions constitute the signal inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we also suggest that repetitive sequence elements of promoter DNA modulate the rigidity of its double-stranded form, which profoundly influences the reaction coordinates of the productive initiation via pausing.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Investigations of the modular structure of bacterial promoters

2006 ◽  
Vol 73 ◽  
pp. 1-10 ◽  
Author(s):  
Nora S. Miroslavova ◽  
Stephen J.W. Busby
Keyword(s):  
Rna Polymerase ◽  
Dna Sequence ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Modular Structure ◽  
Bacterial Rna ◽  
Sequence Elements ◽  
Promoter Dna ◽  
Transcript Initiation ◽  
Rna Polymerase Holoenzyme

Bacterial RNA polymerase holoenzyme carries different determinants that contact different promoter DNA sequence elements. These contacts are essential for the recognition of promoters prior to transcript initiation. Here, we have investigated how active promoters can be built from different combinations of elements. Our results show that the contribution of different contacts to promoter activity is critically dependent on the overall promoter context, and that certain combinations of contacts can hinder transcription initiation.

scholarly journals A TBP-independent mechanism for RNA Polymerase II transcription

2021 ◽  
Author(s):  
James Z.J. Kwan ◽  
Thomas F. Nguyen ◽  
Marek A. Budzyński ◽  
Jieying Cui ◽  
Rachel M. Price ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Embryonic Stem ◽  
Promoter Regions ◽  
Pol Ii ◽  
Promoter Dna ◽  
Tata Box Binding Protein

AbstractTranscription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the Pre-Initiation Complex onto promoter DNA. Decades of in vitro and yeast research have shown that the TATA-box binding protein (TBP) is essential to Pol II initiation by triggering the binding of other general transcription factors, and ensuring proper Pol II loading. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells (mESCs) has no global effect on ongoing Pol II transcription. Surprisingly, Pol II transcriptional induction through the Heat Shock Response or cellular differentiation also occurs normally in the absence of TBP. In contrast, acute TBP depletion severely impairs initiation by RNA Polymerase III. Lastly, we show that a metazoan-specific paralog of TBP is expressed in mESCs and that it binds to promoter regions of active Pol II genes even in the absence of TBP. Taken together, our findings reveal an unexplored TBP-independent process in mESCs that points to a diversity in Pol II transcription initiation mechanisms.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

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